骨形态发生蛋白2与壳聚糖神经支架复合体修复兔面神经损伤

Repair of rabbit facial nerve with the combined use of bone morphogenetic protein-2 and chitosan nerves scaffolds

目的:应用生物可降解材料构成的导管通过载体与外源性骨形态发生蛋白(bonemorphogeneticprotein,BMP-2)相结合,构筑新型具有生物和人工合成材料优势的神经支架——组织工程复合体,探讨其对兔面神经损伤的修复。方法:实验于2007-01/06在兰州大学动物实验中心完成。①自制BMP-2及壳聚糖神经支架和单纯壳聚糖神经支架。②暴露兔面神经上颊支,切除长2mm神经,任其回缩,造成8mm神经缺损的动物模型。③选择新西兰白兔24只,按随机数字表法分为2组,复合神经支架组和单纯壳聚糖神经支架组,每组12只。同时采用自身左右对照,设为对照组(自体神经离断后倒置,吻合于神经缺损处)。④一般观察:肉眼观察兔双侧颊肌萎缩程度及活动度。⑤形态学检查:术后16,30周,切取上颊支神经干,近侧吻合口纵切面行苏木精-伊红染色,观察神经再生情况;术后30周,透射电镜下观察再生神经超微结构;术后16,30周,用德国IBAS-I+Ⅱ型计算机图像处理系统作图像处理,计算神经纤维总数和纤维直径、轴突直径及髓鞘厚度。结果:纳入动物24只,均进入结果分析。①大体形态:兔面神经上颊支损伤后2周面肌萎缩,复合神经支架组、单纯壳聚糖神经支架组、对照组分别于术后8,9,11周时开始逐渐恢复正常。②术后16周,单纯壳聚糖神经支架组与复合神经支架组光镜下可见神经外膜有新生血管,再生纤维呈束状分布,粗细不均匀,束间有新生血管。神经纤维排列整齐,无神经瘤形成。术后30周,光镜下可见复合神经支架组神经密集程度、血管化和髓鞘化程度优于单纯壳聚糖神经支架组。对照组形成的神经纤维束较为分散,不均匀,髓鞘处组织染色较差。③术后16,30周兔面神经再生神经图像分析结果:神经纤维的排列、血管化程度、直径和数目、髓鞘壁、轴突形状等各检测指标均优于单纯壳聚糖神经支架组。④术后30周,透射电镜下复合神经支架组神经再生超微形态学更接近对照组,优于单纯壳聚糖神经支架组。结论:可吸收性BMP-2与壳聚糖神经支架复合体能有效地引导兔面神经再生并恢复其功能,优于单纯壳聚糖神经支架。

AIM： Pipes consisting of biodegradable materials combined with exogenous bone morphogenetic protein （BMP）-2 were used to construct a new nerve scaffolds tissue engineered composite that possesses biological and artificial synthetic advantage, and repairing rabbit facial nerve injury was explored. METHODS： The experiment was carried out in the animal test center of Lanzhou University from January to June in 2007.①The BMP-2 and the chitosan nerve scaffolds were prepared.②A 8-ram facial nerve injury model was induced in rabbits by removing a 2-mm nerve in upper-cheek branches of facial nerve.③Totally 24 New Zealand white rabbits were divided into 2 groups at random： compound nerve scaffold group and chitosan nerve scaffold group, each group containing 12 animals. Meanwhile self-controlled observation was performed as the control group （the isolated autologous nerves were placed invertedly and anastomosed on the nerve defect area）.④Amyotrophy and activity of rabbit bilateral cheek muscles were observed macroscopically.⑤Upper-cheek branches of facial nerve were obtained in postoperative 16 and 30 weeks. Proximal anastomotic stroma was stained with hematoxylin-eosin along longitudinal section to observe the nerve regeneration. The ultra-structure of regenerative nerves was detected under transmission electron microscope at 30 weeks. IBAS-Ⅰ＋ Ⅱ type computer image processing system （Germany） was used at 16 and 30 weeks to count the total nerve fibers and calculate the fiber diameter, axonal diameter and myelin sheath thickness. RESULTS： Twenty-four rabbits were all involved in the result analysis.①Facial amyotrophy occurred 2 weeks after upper-cheek branch injury of rabbit facial nerves. The recovery in compound nerve scaffold group, chitosan nerve scaffold group and control group appeared 8, 9 and 11 weeks, respectively.②At postoperative 16 weeks, new vessels were visible in epineurium of chitosan nerve scaffold group and compound scaffold group, and regenerative fibers were uneven in sarciniform. Nerve fibers arranged in order without neuroma formation. At postoperative 30 weeks, the nerve density, vascularization and myelinization of compound scaffold group were all better than those in chitosan scaffold group. In the control group, the nerve fiber bundles were scattered and uneven, with weak staining in the myelin sheath.③The results of image analysis for rabbit facial nerve regeneration at postoperative 16 and 30 weeks revealed that, nerve tYlbers arrangement, vascularization, diameter and count, myelin sheath wall, and axonal shape of compound scaffold group were all superior to those in chitosan scaffold group.④Under transmission electron microscope, the ultra microstructure of the regenerative nerves in composite scaffolds were even more closer to the control group and significantly superior to that in chitosan scaffolds at 30 weeks. CONCLUSION： The absorbable BMP-2 and the chitosan nerve scaffolds, surpassing pure chitosan nerve scaffold, can guide the rabbit facial nerve regenerate and recover its function effectively.